1. Field of the Invention
The present invention is directed to the field of molecular genetics of cancer. Specifically, the present invention relates to human lymphomas in which a translocation between chromosomes 2 and 5 (referred to in the art as xe2x80x9ct(2;5)xe2x80x9d) has occurred. On a molecular level, the DNA rearrangement in t(2;5) results in the fusion of the known NPM gene with a novel gene named ALK (Anaplastic Lymphoma Kinase) that encodes a protein tyrosine kinase (PTK).
2. Related Art
Chromosomal abnormalities are frequently associated with malignant diseases. In a number of instances, specific chromosomal translocations have been characterized, which generate fusion genes encoding proteins with oncogenic properties (Sawyers et al., Cell 64:337-350 (1991)). Perhaps the best example of genetic characterization of a malignant disease is provided by the analysis of the chromosomal abnormalities unique to different subsets of non-Hodgkin""s lymphoma (NHL). The NHL subset commonly referred to as large cell lymphoma (which comprises xcx9c25% and 40% of NHL in children and adults, respectively) has historically been the most ill-defined because of its marked cytological, immunological and clinical heterogeneity.
Approximately one-third of large cell lymphomas (10% of all NHL) contain the t(2;5)(p23;q35), usually as the only cytogenetic abnormality (R. Rimokh et al., Br. J. Haematol. 71:31-36 (1989); D. Mason et al., Br. J. Haematol. 74:161-168 (1990); H. Stein and F. Dallenbach, in Neoplastic Hematopathology, D. M. Knowles, ed., Williams and Wilkins, Baltimore (1992), pp. 675-714), suggesting that rearrangement of cellular proto-oncogenes on these chromosomes contributes to lymphomagenesis.
The majority of t(2;5)-positive lymphomas (70-75%) express T-lymphoid markers, although usually in the aberrant, incomplete fashion characteristic of T-cell malignancies (e.g., preservation of CD2 and CD4 with infrequent expression of CD3) (M. E. Kadin, J. Clin. Oncol. 12:884-887 (1994); J. T. Sandlund et al., Blood 84:2467-2471 (1994)). Less commonly, these neoplasms bear B-cell markers (xcx9c15%) or have a null phenotype with neither B- nor T-antigen expression (10%). Lymphomas with the t(2;5) typically involve lymph nodes, skin, lung, soft tissue, bone and the gastrointestinal tract, and arise predominantly from activated T lymphocytes (Y. Kaneko et al., Blood 73:806-813 (1989); M. M. Le Beau et al., Leukemia 3:866-870 (1989); R. Rimokh et al., Br. J. Haematol. 71:31-36 (1989); D. Y. Mason et al., Br. J. Haematol. 74:161-168 (1990); M. A. Bitter Am. J. Surg. Pathol. 14:305-316 (1990); M. E. Kadin, J. Clin. Oncol. 9:533-536 (1991); J. P. Greer et al., J. Clin. Oncol. 9:539-547 (1991); V. Vecchi et al., Med. Pediatr. Oncol. 21:402-410 (1993)). The malignant cells express IL-2 receptors and CD30 (Ki-1) antigen, a receptor for a newly described member of the tumor necrosis factor ligand family (H. Durkop et al., Cell 68:421-427 (1992); C. A. Smith et al., Cell 73:1349-1360 (1993)). By the updated Kiel lymphoma classification, most tumors with the t(2;5) are classified as anaplastic large cell non-Hodgkin""s lymphomas (A. G. Stansfeld et al., Lancet 1:292-293 (1988)). These tumors typically behave as aggressive, high-grade NHL with most patients having advanced stage disease at presentation. From 30% to 40% of patients eventually succumb to their disease despite aggressive therapeutic intervention.
Disclosed herein is the cloning and sequencing of human nucleic acid sequences which are rearranged in the t(2;5)(p23;q35) chromosomal translocation event which occurs in human t(2;5) lymphoma. The rearrangement was found to bring sequences from the nucleolar phosphoprotein gene (the NPM gene) on chromosome 5q35 to those from a previously unidentified protein tyrosine kinase (PTK) gene (the ALK gene) on chromosome 2p23. The sequence of the novel ALK gene and ALK protein, as well as the sequence of the t(2;5) fusion gene and fusion protein (the NPM/ALK gene and NPM/ALK protein, respectively), are disclosed herein.
The NPM gene encodes a highly conserved nonribosomal RNA-binding protein that shuttles ribosomal ribonucleoproteins (rRNPs) between the nucleolus and the cytoplasm; rRNPs associate with NPM in the nucleolus, are carried to the cytoplasm, and are released at the maturing ribosomes (M. S. Schmidt-Zachmann et al., EMBO J. 6:1881-1890 (1987); M. S. Schmidt-Zachmann et al., Chromosoma. 96:417-426 (1988)). Bidirectional movement of NPM between the nucleolus and cytoplasm has been elegantly demonstrated in studies monitoring the equilibration of the protein between nuclei present in chicken-mouse heterokaryons (R. A. Borer et al., Cell 56:379-390 (1989)). Several groups have shown that NPM can exist in the cell as either a monomer or a homo-oligomeric hexamer, associated in a head-to-head/tail-to-tail fashion; it is not clear which form of the protein moves between the nucleolus and cytoplasm (M. S. Schmidt-Zachmann et al., Chromosoma. 96:417-426 (1988); B. Y. Yung et al, Biochim. Biophys. Acta. 925:74-82 (1989); Q. R. Liu et al., Eur. J. Biochem. 200:715-721 (1991)).
The NPM/ALK fusion gene was initially identified in anaplastic large cell lymphomas. However, its presence has since been observed in a significant number of diffuse and immunoblastic large cell cases as well. Expression of the NPM/ALK fusion gene in lymphoid cell lines which are otherwise dependent on IL-3 for growth results in transformed cells which proliferate in an IL-3-independent manner. This result suggests a means by which the NPM/ALK fusion acts to promote tumorigenesis and provides for methods of culturing lymphoid cells in vitro in the absence of IL-3.
Utilizing the sequences of the NPM/ALK fusion gene, the present invention provides methods of identifying the presence of nucleic acids containing the NPM/ALK fusion by means such as nucleic acid hybridization and detection methods (e.g., xe2x80x9cSoutherns,xe2x80x9d xe2x80x9cNorthernsxe2x80x9d and the like) fluorescence in situ hybridization (FISH) and detection methods, or polymerase chain reaction (PCR) amplification and detection methods. Such methods can be used in, inter alia, to determine if particular cells or tissues express ALK or NPM/ALK coding sequences, or diagnostic assays designed to determine, for example, if a mammal has cancer or a genetic predisposition to (i.e., is at an increased risk of developing) cancer.
Detection methods utilizing the ALK sequences of the invention as probes are further used to isolate and clone ALK sequences and genes from a variety of mammalian species, and the ALK sequences and genes so prepared provide the foundation for further embodiments of the invention. For example, a xe2x80x9cSouthernxe2x80x9d assay is used to identify clones from a library of murine cDNA sequences using human ALK DNA sequences as a radiolabeled probe. These ALK-positive clones are isolated, and the nucleotide sequence of the mouse cDNA inserted into the vector in these clones is determined using any standard method of nucleotide sequencing known to those of skill in the art. The human and murine ALK nucleotide sequences are aligned by computer program or by hand, and regions of identical or, at least well-conserved, nucleotide sequence between the ALK genes of the two species are identified. These identical/conserved sequences are used to design oligonucleotides which function as ALK-specific primers to be used in PCR amplifications in which the template DNA is genomic DNA or cDNA from a third mammal (i.e., one which is neither a mouse nor a human) in order to obtain ALK DNA, that can be cloned and sequenced, from said third mammal. Alternatively, human or murine ALK sequences, or oligonucleotides derived therefrom, are labeled and used as probes to identify ALK-positive clones in libraries prepared from genomic DNA or cDNA from a third mammal. In like fashion, ALK sequences from any mammal can be prepared and tested for its use in any appropriate embodiment of the present invention. Furthermore, cDNAs derived from alternatively-spliced human ALK transcripts (see Example 2(B)) are prepared by reverse transcription, cloning and identification by hybridization according to methods known in the art (see, for example, Chapters 7-8 in Sambrook et al., eds., Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
The nucleotide sequences of the ALK, as well as the NPM/ALK, genes of the invention are also utilized to design and prepare agents which specifically inhibit the expression of the ALK and/or NPM/ALK genes in cells for therapeutic and other purposes. For example, antisense oligonucleotides and ribozymes specific for ALK and/or NPM/ALK are prepared using the nucleotide sequences of the invention.
The ALK and NPM/ALK genes of the invention are further utilized in methods of producing ALK or NPM/ALK proteins, respectively, by introduction of the appropriate gene into a host/vector expression system. Of course, ALK and NPM/ALK proteins or polypeptides derived therefrom may also be produced by other means known in the art such as, for example, chemical synthesis or in vitro transcription/translation.
The ALK and NPM/ALK proteins and polypeptide sequences of the invention, whether produced by host/vector systems or otherwise, can be used to produce antibodies which (a) specifically recognize (i.e., bind) the NPM/ALK protein, (b) specifically recognize the ALK protein or (c) specifically recognize both the ALK and NPM/ALK proteins.
The present invention further provides methods of detecting the presence of the ALK and/or NPM/ALK proteins which are based on antibody detection systems. For example, because the NPM/ALK fusion protein is expressed in t(2;5) lymphoma cells, but the normal ALK protein is not, antibodies which recognize the fusion protein can be used to detect the presence of the NPM/ALK fusion protein in a sample containing such cells, or to detect the presence of t(2;5) lymphoma cells in a tissue sample suspected of containing such cells.
The invention further provides compartmentalized kits to receive in close confinement one or more containers containing the reagents used in one or more of the above described detection methods.
The present invention further provides methods for isolating and identifying the natural ligand(s) bound by the NPM/ALK or ALK proteins, and for identifying derivatives of the ligand(s) that act to inhibit the action of the NPM/ALK and/or ALK proteins.
Finally, the present invention provides for transgenic animals, preferably mice, which (a) lack a functional copy of the endogenous ALK gene (e.g., ALK xe2x80x9cknockoutxe2x80x9d mice), (b) contain and express an NPM/ALK fusion protein derived from an exogenous source and subsequently introduced into the genome of the animal or (c) both lack a functional ALK gene and express an introduced NPM/ALK fusion gene. Methods of utilizing such mice to identify and test carcinogenic or therapeutic compositions are also described herein.